Seiji Shibasaki

Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses.

Abstract. The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as α-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 104–105 molecules of α-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

Abstract An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration.

Abstract. We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.

Development of combinatorial bioengineering using yeast cell surface display—order-made design of cell and protein for bio-monitoring

A genetic system to display proteins as their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae, has been exploited. Surface-engineered (arming) cells displaying amylase or cellulase could assimilate starch or cellulose as the sole carbon source, although S. cerevisiae can not intrinsically assimilate them. Arming cells with a green fluorescent protein (GFP) from Aequorea victoria can emit green fluorescence from the cell surface in response to the environmental conditions. From these results, we attempted to construct a system to monitor the foreign protein production in yeast by simultaneous displaying the enhanced GFP (EGFP). The expression in yeast of the Escherichia coli beta-galactosidase-encoding gene was examined as an example of intracellular production and that of the human interferon-alpha (omega, IFN-omega)-encoding gene as an example of extracellular production. Their productions and the simultaneous surface-display of EGFP as a reporter were controlled by the same promoter, GAL1. The relationship among fluorescence signals and their productions was evaluated. The surface-display system, unlike one using tag-proteins, would be able to facilitate the monitoring of native protein productions in bioprocesses using living cells in real time by the combination of promoters and GFP variants.

An arming yeast with the ability to entrap fluorescent 17β-estradiol on the cell surface

Abstract. We constructed a novel surface-engineered yeast displaying the ligand-binding domain of the rat estrogen receptor (ERLBD). ERLBD, display of which on the yeast cell surface was confirmed by immunofluorescence, possessed strong binding activity to fluorescent 17β-estradiol – an analogue of the natural ligand of the estrogen receptor – that was comparable to the activity of the native receptor. Environmental homeostasis has recently been disturbed by endocrine disruptors, which cause confusion in the hormone secretion system. It is therefore very important to identify chemical compounds with hormone-like activity and remove them from the environment. The present results demonstrate that the new arming yeast displaying ERLBD on its cell surface will be capable of screening, entrapping, and removing estradiol-like compounds from the environment.